Amylolytic enzyme variants

ABSTRACT

The inventors have discovered some striking, and not previously predicted structural similarities and differences between the structure of Novamyl and the reported structures of CGTases, and based on this they have constructed variants of maltogenic alpha-amylase having CGTase activity and variants of CGTase having maltogenic alpha-amylase activity. Further, on the basis of sequence homology between Novamyl® and CGTases, the inventors have constructed hybrid enzymes with one or more improvements to specific properties of the parent enzymes, using recombinant DNA methodology.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. Ser. No. 09/645,707,filed on Aug. 24, 2000 (now allowed), which is a continuation ofPCT/DK/99/00087, filed on Feb. 26, 1999, and claims priority under 35U.S.C. 119 of Danish application nos. PA 1998 00269 and PA 1998 00273,both filed on Feb. 27, 1998, and U.S. provisional application Nos.60/077,509 and 60/077,795, filed on Mar. 11, 1998 and Mar. 12, 1998,respectively, the contents of which are fully incorporated herein byreference.

FIELD OF THE INVENTION

[0002] The present invention relates to methods of converting amaltogenic alpha-amylase into a cyclodextrin glucanotransferase (CGTase)or vice versa or creating hybrids of the two. The invention also relatesto the variants made by the methods.

BACKGROUND OF THE INVENTION

[0003] Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) andmaltogenic alpha-amylase (EC 3.2.1.133) are two classes of glycosylasesthat degrade starch by hydrolysis of the α-(1,4)-glycosidic bonds, butthe initial products are predominantly cyclic for CGTases and linear forthe maltogenic alpha-amylase.

[0004] Cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19), alsodesignated cyclodextrin glucanotransferase or cyclodextringlycosyltransferase, abbreviated herein as CGTase, catalyses theconversion of starch and similar substrates into cyclomaltodextrins viaan intramolecular transglycosylation reaction, thereby formingcyclomaltodextrins (or CD) of various sizes. Commercially most importantare cyclodextrins of 6, 7 and 8 glucose units, termed α-, β- andγ-cyclodextrins, respectively.

[0005] CGTases are widely distributed and from several differentbacterial sources, including Bacillus, Brevibacterium, Clostridium,Corynebacterium, Klebsiella, Micrococcus, Thermoanaerobacter andThermoanaerobacterium have been extensively described in the literature.A CGTase produced by Thermoanaerobacter sp. has been reported in NormanB E, Jørgensen S T; Denpun Kaqaku 1992 39 99-106, and WO 89/03421, andthe amino acid sequence has been disclosed in WO 96/33267. The sequenceof CGTases from Thermoanaerobacterium thermosulfurigenes and fromBacillus circulansis available on the Internet (SCOP or PDF home pages)as pdf file 1 CIU, and the sequence of a CGTase from B. circulans isavailable as pdf file 1 CDG.

[0006] Tachibana, Y., Journal of Fermentation and Bioengineering, 83(6), 540-548 (1997) describes the cloning and expression of a CGTase.Variants of CGTases have been described by Kim, Y. H., Biochemistry andMolecular Biology International, 41 (2), 227-234 (1997); Sin K-A,Journal of Biotechnology, 32 (3), 283-288 (1994); D Penninga,Biochemistry, 34 (10), 3368-3376 (1995); and WO 96/33267.

[0007] Maltogenic alpha-amylase (glucan 1,4-a-maltohydrolase, E.C.3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose inthe alpha-configuration, and is also able to hydrolyze maltotriose aswell as cyclodextrin.

[0008] A maltogenic alpha-amylase from Bacillus (EP 120 693) iscommercially available under the trade name Novamyl® (product of NovoNordisk A/S, Denmark) and is widely used in the baking industry as ananti-staling agent due to its ability to reduce retrogradation of starch(WO 91/04669).

[0009] The maltogenic alpha-amylase Novamyl® shares severalcharacteristics with cyclodextrin glucanotransferases (CGTases),including sequence homology (Henrissat B, Bairoch A; Biochem. J., 316,695-696 (1996)) and formation of transglycosylation products(Christophersen, C., et al., 1997, Starch, vol. 50, No.1, 39-45).

BRIEF DESCRIPTION OF THE INVENTION

[0010] The inventors have discovered some striking, and not previouslypredicted structural similarities and differences between the structureof Novamyl and the reported structures of CGTases, and based on thisthey have constructed variants of maltogenic alpha-amylase having CGTaseactivity and variants of CGTase having maltogenic alpha-amylaseactivity. Further, on the basis of sequence homology between Novamyl®and CGTases, the inventors have constructed hybrid enzymes with one ormore improvements to specific properties of the parent enzymes, usingrecombinant DNA methodology.

[0011] Accordingly, the present invention provides a polypeptide which:

[0012] a) has at least 70% identity to amino acids 1-686 of SEQ ID NO:1;

[0013] b) comprises an amino acid modification which is an insertion,substitution or deletion compared to SEQ ID NO: 1 in a regioncorresponding to amino acids 40-43, 78-85, 136-139, 173-180,188-195 or259-268; and

[0014] c) has the ability to form cyclodextrin when acting on starch.

[0015] The invention also provides a polypeptide which:

[0016] a) has an amino acid sequence having at least 70% identity to aparent cyclodextrin glucanotransferase (CGTase);

[0017] b) comprises an amino acid modification which is an insertion,substitution or deletion compared to the parent CGTase in a regioncorresponding to amino acids 40-43, 78-85,136-139,173-180,188-195 or259-268 of SEQ ID NO: 1; and

[0018] c) has the ability to form linear oligosaccharides when acting onstarch.

[0019] Further, the invention provides a method for constructing amaltogenic alpha-amylase, comprising:

[0020] a) recombining DNA encoding a cyclodextrin glucanotransferase(CGTase) and DNA encoding a maltogenic alpha-amylase;

[0021] b) using the recombinant DNA to express a polypeptide; and

[0022] c) testing the polypeptide to select a polypeptide having theability to form linear oligosaccharides when acting on starch.

[0023] Finally, the invention provides a method of selecting DNAencoding maltogenic alpha-amylase in a DNA pool, comprising:

[0024] a) amplifying DNA encoding maltogenic alpha-amylase by apolymerase chain reaction (PCR) using primers encoding a partial aminoacid sequence of amino acids 1-686 of SEQ ID NO: 1, preferablycomprising at least 5 amino acid residues, preferably comprising one ormore of positions 188-196, more preferably comprising positions 190-194,

[0025] b) cloning and expressing the amplified DNA, and

[0026] c) screening for maltogenic alpha-amylase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 shows plasmid pCA31, described in Example 1.

[0028]FIG. 2 is a diagram showing the shuffling of Novamyl with CGTases,as described in Example 3.

[0029]FIG. 3 is a diagram showing the selection of clones with desiredfeatures by PCR, as described in Examples 2 and 3.

[0030]FIGS. 4a-4 b shows an alignment of the amino acid sequence ofNovamyl (1-686 of SEQ ID NO: 1) with the sequence of 3 CGTases asdescribed below.

DETAILED DESCRIPTION OF THE INVENTION Maltogenic Alpha-amylase

[0031] The parent maltogenic alpha-amylase used in the invention is anenzyme classified in EC 3.2.1.133. The enzymatic activity does notrequire a non-reducing end on the substrate and the primary enzymaticactivity results in the degradation of amylopectin and amylose tomaltose and longer maltodextrins. It is able to hydrolyze amylose andamylopectin to maltose in the alpha-configuration, and is also able tohydrolyze maltotriose as well as cyclodextrin.

[0032] A particularly preferred maltogenic alpha-amylase is the amylasecloned from Bacillus as described in EP 120 693 (hereinafter referred toas Novamyl). Novamyl has the amino acid sequence set forth in aminoacids 1-686 of SEQ ID NO: 1. Novamyl is encoded in the gene harbored inthe Bacillus strain NCIB 11837 which has the nucleic acid sequence setforth in SEQ ID NO:1.

CGTase

[0033] The parent CGTase used in the invention is an enzyme classifiedin EC 2.4.1.19. It may be from any source, e.g. bacterial sources,including Bacillus, Brevibacterium, Clostridium, Corynebacterium,Klebsiella, Micrococcus, Thermoanaerobacter and Thermoanaerobacterium.

[0034] The CGTase preferably has one or more of the followingcharacteristics:

[0035] i) an amino acid sequence having at least 50% identity to aminoacids 1-686 of SEQ ID NO: 1, preferably at least 60%;

[0036] ii) being encoded by a DNA sequence which hybridizes atconditions described below to the DNA sequence set forth in SEQ ID NO:1or to the DNA sequence encoding Novamyl harbored in the Bacillus strainNCIB 11837; and

[0037] iii)a catalytic binding site comprising amino acid residuescorresponding to D228, E256 and D329 as shown in the amino acid sequenceset forth in amino acids 1-686 of SEQ ID NO: 1.

Variants of CGTase

[0038] The CGTase variant of this invention has the ability to formlinear oligosaccharides when acting on starch. The starch hydrolysis andthe analysis of initial reaction products may be carried out asdescribed in an Example.

[0039] The CGTase variant has a modification of at least one amino acidresidue in a region corresponding to residues 40-43, 78-85, 136-139,173-180, 189-195 or 259-268 of SEQ ID NO: 1. Each modification may be aninsertion, a deletion or a substitution, of one or more amino acidresidues in the region indicated. The modification of the parent CGTaseis preferably such that the resulting modified amino acid or amino acidsequence more closely resembles the corresponding amino acid orstructural region in Novamyl. Thus, the modification may be an insertionof or a substitution with an amino acid present at the correspondingposition of Novamyl, or a deletion of an amino acid not present at thecorresponding position of Novamyl.

[0040] The CGTase variant may particularly comprise an insertion into aposition corresponding to the region D190-F194 of Novamyl (amino acidsequence shown in SEQ ID NO: 1). The insertion may comprise 3-7 aminoacids, particularly 4-6, e.g. 5 amino acids. The insertion may beDPAGF(SEQ ID NO:27) as found in Novamyl or an analogue thereof, e.g.with the first amino acid being negative, the last one being aromatic,and the ones in between being preferably P, A or G. The variant mayfurther comprise a substitution at the position corresponding to T189 ofNovamyl with a neutral amino acid which is less bulky than F, Y or W.Other examples of insertions are DAGF(SEQ ID NO:28), DPGF(SEQ ID.NO:29), DPF, DPAAGF(SEQ ID NO:30), and DPAAGGF(SEQ ID NO:31).

[0041] Modifications in the region 78-85 preferably include deletion of2-5 amino acids, e.g. 3 or 4. Preferably, any aromatic amino acid in theregion 83-85 should be deleted or substituted with a non-aromatic.

[0042] Modifications in the region 259-268 preferably include deletionof 1-3 amino acid, e.g. two. The region may be modified so as tocorrespond to Novamyl

[0043] The CGTase variant may comprise further modifications in otherregions, e.g. regions corresponding to amino acids 37-39, 44-45, 135,140-145, 181-186, 269-273, or 377-383 of Novamyl.

[0044] Additional modifications of the amino acid sequence may bemodeled on a second CGTase, i.e. an insertion of or substitution with anamino acid found at a given position in the second CGTase, or they maybe made close to the substrate (less than 8 Å from the substrate, e.g.less than 5 Å or less than 3 Å) as described in WO 96/33267.

[0045] The following are some examples of variants based on a parentCGTase from Thermoanaerobacter (using B. circulans numbering). Similarvariants may be made from other CGTases.

[0046] L194F+*194aT+*194bD+*194cP+*194dA+*194eG+D196S

[0047]L87H+D89*+T91G+F91aY+G92*+G93*+S94*+L194F+*194aT+*194bD+*194cP+*194dA+*194eG+D196S

[0048] *194aT+*194bD+*194cP+*194dA+*194eG+D196S

[0049] L87H+D89*+T91 G+F91aY+G92*+G93*+S94*+*194aT+*194bD+*194cP+*194dA+*194eG+D196S

[0050] Y260F+L261G+G262D+T263D+N264P+E265G+V266T+*266aA+*266bN+D267H+P268V

[0051]*194aT+*194bD+*194cP+*194dA+*194eG+D196S+Y260F+L261G+G262D+T263D+N264P+E265G+V266T+*266aA+*266bN+D267H+P268V

Variants of Novamyl

[0052] The Novamyl variant of this invention has the ability to formcyclodextrin when acting on starch. The starch hydrolysis and theanalysis of reaction products may be carried out as described in anExample.

[0053] The Novamyl variant has a modification of at least one amino acidresidue in the same regions described above for CGTase variants.However, the modifications are preferably in the opposite direction,i.e. such that the resulting modified amino acid or amino acid sequencemore closely resembles the corresponding amino acid or structural regionof a CGTase. Thus, the modification may be an insertion of or asubstitution with an amino acid present at the corresponding position ofa CGTase, or a deletion of an amino acid not present at thecorresponding position of a CGTase.

[0054] Preferred modifications include a deletion in the region 190-195,preferably the deletion Δ (191-195) and/or a substitution of amino acid188 and/or 189, preferably F188L and/or Y189Y.

Amino Acid Identity

[0055] For purposes of the present invention, the degree of identity maybe suitably determined according to the method described in Needleman,S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48,443-45, with the following settings for polypeptide sequence comparison:GAP creation penalty of 3.0 and GAP extension penalty of 0.1. Thedetermination may be done by means of a computer program known such asGAP provided in the GCG program package (Program Manual for theWisconsin Package, Version 8, August 1994, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711).

[0056] The variants of the invention have an amino acid identity withthe parent enzyme (Novamyl or CGTase) of at least 70%, preferably atleast 80%, e.g. at least 90%, particularly at least 95% or at least 98%.

Hybridization

[0057] The hybridization referred to above indicates that the analogousDNA sequence hybridizes to the nucleotide probe corresponding to theprotein encoding part of the nucleic sequence shown in SEQ ID NO:1,under at least low stringency conditions as described in detail below.

[0058] Suitable experimental conditions for determining hybridization atlow stringency between a nucleotide probe and a homologous DNA or RNAsequence involves presoaking of the filter containing the DNA fragmentsor RNA to hybridize in 5×SSC (sodium chloride/sodium citrate, Sambrook,J., Fritsch, E. J., and Maniatis, T. (1989) Molecular cloning: alaboratory manual, Cold Spring Harbor Laboratory Press, New York) for 10min, and prehybridization of the filter in a solution of 5×SSC,5×Denhardt's solution (Sambrook, et al., op.cit.), 0.5% SDS and 100μg/ml of denatured sonicated salmon sperm DNA (Sambrook, et al.,op.cit.), followed by hybridization in the same solution containing arandom-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem.132:6-13), ³²P-dCTP-labeled (specific activity >1×10⁹ cpm/μg) probe for12 hours at ca. 45° C. The filter is then washed twice for 30 minutes in2×SSC, 0.5% SDS at least 55° C. (low stringency), more preferably atleast 60° C. (medium stringency), more preferably at least 65° C.(medium/high stringency), more preferably at least 70° C. (highstringency), even more preferably at least 75° C. (very highstringency).

[0059] Molecules which hybridize to the oligonucleotide probe underthese conditions are detected by exposure to x-ray film.

Corresponding Amino Acids

[0060] Corresponding amino acids for the following 4 amino acidsequences are shown in the alignment in FIGS. 4a-4 b which is based onthe three-dimensional structure of the sequences.

[0061] 1) Novamyl (amino acids 1-686 of SEQ ID NO: 1)

[0062] 2) CGTase from Thermoanaerobacterium thermosulfurigenes (pdf file1 CIU)

[0063] 3) CGTase from Thermoanaerobacter, described in WO 96/33267

[0064] 4) CGTase from Bacillus circulans (pdf file 1 CDG)

[0065] Corresponding amino acid residues in other CGTases may be foundby aligning with one of the sequences in FIGS. 4a-4 b by to the methoddescribed in Needleman (supra) using the same parameters, e.g. by meansof the GAP program (supra).

Nomenclature for Amino Acid Modifications

[0066] The nomenclature used herein for defining mutations isessentially as described in WO 92/05249. Thus, F188L indicates asubstitution of the amino acid F (Phe) in position 188 with the aminoacid L (Leu). Δ (191-195) or Δ (191-195) indicates a deletion of aminoacids in positions 191-195. 192-A-193 indicates an insertion of Abetween amino acids 192 and 193. *194aT indicates an insertion of T atthe first position after 194. G92* indicates a deletion of G at position92.

Recombination of CGTase and Maltogenic Alpha-amylase

[0067] The present invention further relates to a method forconstructing a variant enzyme comprising Novamyl and one or more parentCGTases, wherein said variant has at least one altered property relativeto Novamyl and said parent CGTases, which method comprises:

[0068] i)generating DNA fragments encoding amino acid sequencesobtainable from Novamyl and said parent CGTases;

[0069] ii)constructing a hybrid variant which contains amino acidsequences generated in step i) by in vivo or in vitro DNA shuffling; and

[0070] iii)testing the resulting variant for said property.

[0071] The methods for generating DNA fragments referred to in step i)of the method above are well known in the art and may include, forexample, treatment of a DNA sequence encoding an amino acid sequencewith a restriction enzyme, e.g., DNAse I.

[0072] The DNA shuffling referred to in step ii) in the method above maybe recombination, either in vivo or in vitro, of nucleotide sequencefragment(s) between two or more polynucleotides resulting in outputpolynucleotides (i.e., polynucleotides having been subjected to ashuffling cycle) having a number of nucleotide fragments exchanged, incomparison to the input polynucleotides (i.e. starting pointpolynucleotides). Shuffling may be accomplished either in vitro or invivo by recombination within a cell by methods described in the art(cf., Crameri, et al, 1997, Nature Biotechnology Vol.15:436-438).

[0073] In a preferred embodiment, at least one DNA fragment obtainablefrom Novamyl in step i) of the method above encodes an amino acidsequence, which is determined to be of relevance for altering saidproperty.

[0074] In a more preferred embodiment, a hybrid variant of a parentCGTase is obtained by the above method comprising a modification of atleast one amino acid residue in the group consisting of amino acidresidues corresponding to residues 37 to 45, residues 135 to 145,residues 173 to 180, residues 189 to 196, residues 261 to 266, residues327 to 330, and residues 370 to 376 of SEQ ID NO: 1.

[0075] In another more preferred embodiment, a hybrid variant comprisingNovamyl and one or more parent CGTases is constructed by the abovemethod in which the amino acid sequence of Asp190-Pro191-Ala192-Gly-193-Phe194 corresponding to the positions in theamino acid sequence shown in SEQ ID NO: 1 is inserted into thecorresponding positions in said hybrid, wherein the correspondingpositions is determined on the basis of amino acid sequence alignment.

[0076] In another more preferred embodiment, a hybrid variant comprisingNovamyl and one or more parent CGTase is obtained by the above method inwhich the amino acid sequence of Asp 190-Prol91-Ala192-Gly193-Phe194-Ser 95 corresponding to the positions in theamino acid sequence shown in SEQ ID NO: 1 is inserted into thecorresponding positions in said hybrid, wherein the correspondingpositions is determined on the basis of amino acid sequence alignment.

[0077] It is possible to use the unique active site loop to selecthybrid enzymes with maltogenic alpha-amylase activity from a library ofrandom recombinants. Thus, a maltogenic alpha-amylase and a CGTase maybe randomly recombined, e.g. by the DNA shuffling method of Crameri A,et al., op.cit. Those resulting mutants containing the Novamyl loop maybe selected using PCR, e.g. as described above in the Examples.

[0078] The property to be altered may be substrate specificity,substrate binding, substrate cleavage pattern, specific activity ofcleavage, transglycosylation, and relative activity of cyclization.

[0079] The DNA sequence encoding a parent CGTase to be used in themethods of the invention may be isolated from any cell or microorganismproducing the CGTase in question using methods known in the art.

Cloning a DNA Sequence Encoding a CGTaseCloninq a DNA Sequence Encodingan a-amylasecloning a DNA Sequence Encoding an a-amylaseCloning a DNASequence Encoding an a-amylaseCloning a DNA Sequence Encoding ana-amylase

[0080] The DNA sequence encoding a parent CGTase may be isolated fromany cell or microorganism producing the CGTase in question, usingvarious methods well known in the art, for example, from the Bacillusstrain NCIB 11837.

[0081] First, a genomic DNA and/or cDNA library should be constructedusing chromosomal DNA or messenger RNA from the organism that producesthe CGTase to be studied. Then, if the amino acid sequence of the CGTaseis known, homologous, labeled oligonucleotide probes may be synthesizedand used to identify CGTase-encoding clones from a genomic libraryprepared from the organism in question. Alter natively, a labeledoligonucleotide probe containing sequences homologous to a known CGTasegene could be used as a probe to identify CGTase-encoding clones, usinghybridization and washing conditions of lower stringency.

[0082] Another method for identifying CGTase-encoding clones involvesinserting fragments of genomic DNA into an expression vector, such as aplasmid, transforming maltogenic alpha-amylase negative bacteria withthe resulting genomic DNA library, and then plating the transformedbacteria onto agar containing a substrate for maltogenic alpha-amylase,thereby allowing clones expressing maltogenic alpha-amylase activity tobe identified.

[0083] Alternatively, the DNA sequence encoding the enzyme may beprepared synthetically by established standard methods, e.g. thephosphoroamidite method described by S. L. Beaucage and M. H. Caruthers(1981) or the method described by Matthes et al. (1984). In thephosphoroamidite method, oligonucleotides are synthesized, e.g. in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

[0084] Finally, the DNA sequence may be of mixed genomic and syntheticorigin, mixed synthetic and cDNA origin or mixed genomic and cDNAorigin, prepared by ligating fragments of synthetic, genomic or cDNAorigin, wherein the fragments correspond to various parts of the entireDNA sequence, in accordance with techniques well known in the art. TheDNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or R. K. Saiki et al. (1988).

Random Mutagenesis

[0085] A general approach for modifying proteins and enzymes has beenbased on random mutagenesis, for instance, as disclosed in U.S. Pat. No.4,894,331 and WO 93/01285. For instance, the random mutagenesis may beperformed by use of a suitable physical or chemical mutagenizing agent,by use of a suitable oligonucleotide, or by subjecting the DNA sequenceto PCR generated mutagenesis. Furthermore, the random mutagenesis may beperformed by use of any combination of these mutagenizing agents. Themutagenizing agent may, e.g., be one which induces transitions,transversions, inversions, scrambling, deletions, and/or insertions.

[0086] Examples of a physical or chemical mutagenizing agent suitablefor the present purpose include ultraviolet (UV) irradiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methylhydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodiumbisulphite, formic acid, and nucleotide analogues. When such agents areused, the mutagenesis is typically performed by incubating the DNAsequence encoding the parent enzyme to be mutagenized in the presence ofthe mutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties.

[0087] When the mutagenesis is performed by the use of anoligonucleotide, the oligonucleotide may be doped or spiked with thethree non-parent nucleotides during the synthesis of the oligonucleotideat the positions which are to be changed. The doping or spiking may bedone so that codons for unwanted amino acids are avoided. The doped orspiked oligonucleotide can be incorporated into the DNA encoding themaltogenic alpha-amylase enzyme by any published technique, using e.g.PCR, LCR or any DNA polymerase and ligase as deemed appropriate.

[0088] Preferably, the doping is carried out using “constant randomdoping”, in which the percentage of wild-type and mutation in eachposition is predefined. Furthermore, the doping may be directed toward apreference for the introduction of certain nucleotides, and thereby apreference for the introduction of one or more specific amino acidresidues. The doping may be made, e.g., so as to allow for theintroduction of 90% wild type and 10% mutations in each position. Anadditional consideration in the choice of a doping scheme is based ongenetic as well as protein-structural constraints. The doping scheme maybe made by using the DOPE program (cf., Tomandl, D. et al., 1997,Journal of Computer-Aided Molecular Design 11:29-38; Jensen, L J,Andersen, K V, Svendsen, A, and Kretzschmar, T (1998) Nucleic AcidsResearch 26:697-702) which, inter alia, ensures that introduction ofstop codons is avoided.

[0089] When PCR-generated mutagenesis is used, either a chemicallytreated or non-treated gene encoding a parent CGTase enzyme is subjectedto PCR under conditions that increase the misincorporation ofnucleotides (Deshler 1992; Leung et al., Technique, Vol.1, 1989, pp.11-15).

[0090] A mutator strain of E. coli (Fowler et al., Molec. Gen. Genet.,133, 1974, pp. 179-191), S. cereviseae or any other microbial organismmay be used for the random mutagenesis of the DNA encoding the CGTaseby, e.g., transforming a plasmid containing the parent CGTase into themutator strain, growing the mutator strain with the plasmid andisolating the mutated plasmid from the mutator strain. The mutatedplasmid may be subsequently transformed into the expression organism.

[0091] The DNA sequence to be mutagenized may be conveniently present ina genomic or cDNA library prepared from an organism expressing theparent CGTase. Alternatively, the DNA sequence may be present on asuitable vector such as a plasmid or a bacteriophage, which as such maybe incubated with or otherwise exposed to the mutagenising agent. TheDNA to be mutagenized may also be present in a host cell either by beingintegrated in the genome of said cell or by being present on a vectorharbored in the cell. Finally, the DNA to be mutagenized may be inisolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence.

[0092] In some cases it may be convenient to amplify the mutated DNAsequence prior to expression or screening. Such amplification may beperformed in accordance with methods known in the art, the presentlypreferred method being PCR-generated amplification using oligonucleotideprimers prepared on the basis of the DNA or amino acid sequence of theparent enzyme.

[0093] Subsequent to the incubation with or exposure to the mutagenisingagent, the mutated DNA is expressed by culturing a suitable host cellcarrying the DNA sequence under conditions allowing expression to takeplace. The host cell used for this purpose may be one which has beentransformed with the mutated DNA sequence, optionally present on avector, or one which was carried the DNA sequence encoding the parentenzyme during the mutagenesis treatment. Examples of suitable host cellsare the following: gram positive bacteria such as Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium, Bacillus thuringiensis, Streptomyces lividans orStreptomyces murinus; and gram negative bacteria such as E. coli.

[0094] The mutated DNA sequence may further comprise a DNA sequenceencoding functions permitting expression of the mutated DNA sequence.

DNA Shuffling

[0095] Alternative methods for rapid preparation of modifiedpolypeptides may be prepared using methods of in vivo or in vitro DNAshuffling wherein DNA shuffling is defined as recombination, either invivo or in vitro, of nucleotide sequence fragment(s) between two or morepolynucleotides resulting in output polynucleotides (i.e.,polynucleotides having been subjected to a shuffling cycle) having anumber of nucleotide fragments exchanged, in comparison to the inputpolynucleotides (i.e. starting point polynucleotides). Shuffling may beaccomplished either in vitro or in vivo by recombination within a cellby methods described in the art.

[0096] For instance, Weber et al. (1983, Nucleic Acids Research, vol.11, 5661-5661) describe a method for modifying genes by in vivorecombination between two homologous genes, wherein recombinants wereidentified and isolated using a resistance marker.

[0097] Pompon et al., (1989, Gene 83:15-24) describe a method forshuffling gene domains of mammalian cytochrome P-450 by in vivorecombination of partially homologous sequences in Saccharomycescereviseae by transforming Saccharomyces cereviseae with a linearizedplasmid with filled-in ends, and a DNA fragment being partiallyhomologous to the ends of said plasmid.

[0098] In WO 97/07205 a method is described whereby polypeptide variantsare prepared by shuffling different nucleotide sequences of homologousDNA sequences by in vivo recombination using plasmid DNA as template.

[0099] U.S. Pat. No. 5,093,257 (Genencor Int. Inc.) discloses a methodfor producing hybrid polypeptides by in vivo recombination. Hybrid DNAsequences are produced by forming a circular vector comprising areplication sequence, a first DNA sequence encoding the amino-terminalportion of the hybrid polypeptide, a second DNA sequence encoding thecarboxy-terminal portion of said hybrid polypeptide. The circular vectoris transformed into a rec positive microorganism in which the circularvector is amplified. This results in recombination of said circularvector mediated by the naturally occurring recombination mechanism ofthe rec positive microorganism, which include prokaryotes such asBacillus and E. coli, and eukaryotes such as Saccharomyces cereviseae.

[0100] One method for the shuffling of homologous DNA sequences has beendescribed by Stemmer (Stemmer, (1994), Proc. Natl. Acad. Sci. USA, Vol.91, 10747-10751; Stemmer, (1994), Nature, vol. 370, 389- 391; Crameri A,Dawes G, Rodriguez E Jr, Silver S, Stemmer WPC (1997) NatureBiotechnology Vol. 15, No. 5 pp. 436-438). The method concerns shufflinghomologous DNA sequences by using in vitro PCR techniques. Positiverecombinant genes containing shuffled DNA sequences are selected from aDNA library based on the improved function of the expressed proteins.

[0101] The above method is also described in WO 95/22625 in relation toa method for shuffling homologous DNA sequences. An important step inthe method described in WO 95/22625 is to cleave the homologous templatedouble-stranded polynucleotide into random fragments of a desired sizefollowed by homologously reassembling of the fragments into full-lengthgenes.

Site-directed Mutagenesis

[0102] Once a maltogenic alpha-amylase-encoding DNA sequence has beenisolated, and desirable sites for mutation identified, mutations may beintroduced using synthetic oligonucleotides. These oligonucleotidescontain nucleotide sequences flanking the desired mutation sites; mutantnucleotides are inserted during oligonucleotide synthesis. In a specificmethod, a single-stranded gap of DNA, bridging the maltogenicalpha-amylase-encoding sequence, is created in a vector carrying themaltogenic alpha-amylase gene. Then the synthetic nucleotide, bearingthe desired mutation, is annealed to a homologous portion of thesingle-stranded DNA. The remaining gap is then filled in with DNApolymerase I (Klenow fragment) and the construct is ligated using T4ligase. A specific example of this method is described in Morinaga etal. (1984). U.S. Pat. No. 4,760,025 discloses the introduction ofoligonucleotides encoding multiple mutations by performing minoralterations of the cassette. However, an even greater variety ofmutations can be introduced at any one time by the Morinaga methodbecause a multitude of oligonucleotides, of various lengths, can beintroduced.

[0103] Another method of introducing mutations into a maltogenicalpha-amylase-encoding DNA sequences is described in Nelson and Long,Analytical Biochemistry 180, 1989, pp. 147-151. It involves a 3-stepgeneration of a PCR fragment containing the desired mutation introducedby using a chemically synthesized DNA strand as one of the primers inthe PCR reactions. From the PCR-generated fragment, a DNA fragmentcarrying the mutation may be isolated by cleavage with restrictionendonucleases and reinserted into an expression plasmid.

Localized Random Mutagenesis

[0104] The random mutagenesis may be advantageously localised to a partof the parent CGTase in question. This may, e.g., be advantageous whencertain regions of the enzyme have been identified to be of particularimportance for a given property of the enzyme, and when modified areexpected to result in a variant having improved properties. Such regionsmay normally be identified when the tertiary structure of the parentenzyme has been elucidated and related to the function of the enzyme.

[0105] The localized, or region-specific, random mutagenesis isconveniently performed by use of PCR generated mutagenesis techniques asdescribed above or any other suitable technique known in the art.Alternatively, the DNA sequence encoding the part of the DNA sequence tobe modified may be isolated, e.g., by insertion into a suitable vector,and said part may be subsequently subjected to mutagenesis by use of anyof the mutagenesis methods discussed above.

Expression of Maltogenic Alpha-amylase Variants

[0106] The construction of the variant of interest is accomplished bycultivating a microorganism comprising a DNA sequence encoding thevariant under conditions which are conducive for producing the variant,and optionally subsequently recovering the variant from the resultingculture broth. This is described in detail further below.

[0107] According to the invention, a DNA sequence encoding the variantproduced by methods described above, or by any alternative methods knownin the art, can be expressed, in the form of a protein or polypeptide,using an expression vector which typically includes control sequencesencoding a promoter, operator, ribosome binding site, translationinitiation signal, and, optionally, a repressor gene or variousactivator genes.

[0108] The recombinant expression vector carrying the DNA sequenceencoding an maltogenic alpha-amylase variant of the invention may be anyvector which may conveniently be subjected to recombinant DNAprocedures, and the choice of vector will often depend on the host cellinto which it is to be introduced. Thus, the vector may be anautonomously replicating vector, i.e. a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid, a bacteriophage or anextrachromosomal element, minichromosome or an artificial chromosome.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

[0109] In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding a maltogenic alpha-amylasevariant of the invention, especially in a bacterial host, are thepromoter of the lac operon of E. coli, the Streptomyces coelicoloragarase gene dagA promoters, the promoters of the Bacillus licheniformisα-amylase gene (amyL), the promoters of the Bacillus stearothermophilusmaltogenic amylase gene (amyM), the promoters of the Bacillusamyloliquefaciens α-amylase (amyQ), the promoters of the Bacillussubtilis xyIA and xyIB genes, etc. For transcription in a fungal host,examples of useful promoters are those derived from the gene encoding A.oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. nigerneutral α-amylase, A. niger acid stable α-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase or A. nidulans acetamidase.

[0110] The expression vector of the invention may also comprise asuitable transcription terminator and, in eukaryotes, polyadenylationsequences operably connected to the DNA sequence encoding the maltogenicalpha-amylase variant of the invention. Termination and polyadenylationsequences may suitably be derived from the same sources as the promoter.

[0111] The vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. Examples of suchsequences are the origins of replication of plasmids pUC19, pACYC177,pUB110, pE194, pAMB1 and plJ702.

[0112] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracycline resistance. Furthermore, the vector may compriseAspergillus selection markers such as amdS, argB, niaD and sC, a markergiving rise to hygromycin resistance, or the selection may beaccomplished by co-transformation, e.g. as described in WO 91/17243.

[0113] While intracellular expression may be advantageous in somerespects, e.g. when using certain bacteria as host cells, it isgenerally preferred that the expression is extracellular. In general,the Bacillus α-amylases mentioned herein comprise a preregion permittingsecretion of the expressed protease into the culture medium. Ifdesirable, this preregion may be replaced by a different preregion orsignal sequence, conveniently accomplished by substitution of the DNAsequences encoding the respective preregions.

[0114] The procedures used to ligate the DNA construct of the inventionencoding maltogenic alpha-amylase variant, the promoter, terminator andother elements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambroo, et al.,op.cit.).

[0115] The cell of the invention, either comprising a DNA construct oran expression vector of the invention as defined above, isadvantageously used as a host cell in the recombinant production of amaltogenic alpha-amylase variant of the invention. The cell may betransformed with the DNA construct of the invention encoding thevariant, conveniently by integrating the DNA construct (in one or morecopies) in the host chromosome. This integration is generally consideredto be an advantage as the DNA sequence is more likely to be stablymaintained in the cell. Integration of the DNA constructs into the hostchromosome may be performed according to conventional methods, e.g. byhomologous or heterologous recombination. Alternatively, the cell may betransformed with an expression vector as described above in connectionwith the different types of host cells.

[0116] The cell of the invention may be a cell of a higher organism suchas a mammal or an insect, but is preferably a microbial cell, e.g. abacterial or a fungal (including yeast) cell.

[0117] Examples of suitable bacteria are gram positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gram negative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

[0118] The yeast organism may favorably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g. Saccharomyces cereviseae. Thefilamentous fungus may advantageously belong to a species ofAspergillus, e.g. Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

[0119] In a yet further aspect, the present invention relates to amethod of producing a maltogenic alpha-amylase variant of the invention,which method comprises cultivating a host cell as described above underconditions conducive to the production of the variant and recovering thevariant from the cells and/or culture medium.

[0120] The medium used to cultivate the cells may be any conventionalmedium suitable for growing the host cell in question and obtainingexpression of the maltogenic alpha-amylase variant of the invention.Suitable media are available from commercial suppliers or may beprepared according to published recipes (e.g. as described in cataloguesof the American Type Culture Collection).

[0121] The maltogenic alpha-amylase variant secreted from the host cellsmay conveniently be recovered from the culture medium by well-knownprocedures, including separating the cells from the medium bycentrifugation or filtration, and precipitating proteinaceous componentsof the medium by means of a salt such as ammonium sulfate, followed bythe use of chromatographic procedures such as ion exchangechromatography, affinity chromatography, or the like.

Screening of Variants with Maltogenic Alpha-amylase Activity

[0122] Variants produced by any of the methods described above may betested, either prior to or after purification, for maltogenicalpha-amylase activity, such as amylolytic activity, in a screeningassay which measures the ability of the variant to degrade starch. Thescreening in step 10 in the above-mentioned random mutagenesis method ofthe invention may be conveniently performed by use of a filter assaybased on the following procedure: A microorganism capable of expressingthe variant of interest is incubated on a suitable medium and undersuitable conditions for secretion of the variant, the medium beingcovered with two filters comprising a protein-binding filter placedunder a second filter exhibiting a low protein binding capability. Themicroorganism is grown on the second, top filter. Subsequent to theincubation, the bottom protein-binding filter comprising enzymessecreted from the microorganism is separated from the second filtercomprising the microorganism. The protein-binding filter is thensubjected to screening for the desired enzymatic activity, and thecorresponding microbial colonies present on the second filter areidentified. The first filter used for binding the enzymatic activity maybe any protein-binding filter, e.g., nylon or nitrocellulose. The secondfilter carrying the colonies of the expression organism may be anyfilter that has no or low affinity for binding proteins, e.g., celluloseacetate or Durspore™.

[0123] Screening consists of treating the first filter to which thesecreted protein is bound with a substrate that allows detection of theamylolytic activity. The enzymatic activity may be detected by a dye,fluorescence, precipitation, pH indicator, IR-absorbance or any otherknown technique for detection of enzymatic activity. The detectingcompound may be immobilized by any immobilizing agent e.g. agarose,agar, gelatine, polyacrylamide, starch, filter paper, cloth; or anycombination of immobilizing agents. For example, amylolytic activity canbe detected by Cibacron Red labeled amylopectin, which is immobilized inagarose. Amylolytic activity on this substrate produces zones on theplate with reduced red color intensity.

[0124] To screen for variants with increased stability, the filter withbound maltogenic alpha-amylase variants can be pretreated prior to thedetection step described above to inactivate variants that do not haveimproved stability relative to the parent CGTase. This inactivation stepmay consist of, but is not limited to, incubation at elevatedtemperatures in the presence of a buffered solution at any pH from pH 2to 12, and/or in a buffer containing another compound known or thoughtto contribute to altered stability e.g., surfactants, EDTA, EGTA, wheatflour components, or any other relevant additives. Filters so treatedfor a specified time are then rinsed briefly in deionized water andplaced on plates for activity detection as described above. Theconditions are chosen such that stabilized variants show increasedenzymatic activity relative to the parent after incubation on thedetection media.

[0125] To screen for variants with altered thermostability, filters withbound variants are incubated in buffer at a given pH (e.g., in the rangefrom pH 2-12) at an elevated temperature (e.g., in the range from50°-110° C.) for a time period (e.g., from 1-20 minutes) to inactivatenearly all of the parent CGTase, rinsed in water, then placed directlyon a detection plate containing immobilized Cibacron Red labeledamylopectin and incubated until activity is detectable. Similarly, pHdependent stability can be screened for by adjusting the pH of thebuffer in the above inactivation step such that the parent CGTase isinactivated, thereby allowing detection of only those variants withincreased stability at the pH in question. To screen for variants withincreased calcium-dependent stability calcium chelators, such asethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid(EGTA), is added to the inactivation buffer at a concentration such thatthe parent CGTase is inactivated under conditions further defined, suchas buffer pH, temperature or a specified length of incubation.

[0126] The variants of the invention may be suitably tested by assayingthe starch-degrading activity of the variant, for instance by growinghost cells transformed with a DNA sequence encoding a variant on astarch-containing agarose plate and identifying starch-degrading hostcells as described above. Further testing in regard to alteredproperties, including specific activity, substrate specificity, cleavagepattern, thermoactivation, thermostability, pH dependent activity oroptimum, pH dependent stability, temperature dependent activity oroptimum, transglycosylation activity, stability, and any other parameterof interest, may be performed on purified variants in accordance withmethods known in the art as described below.

[0127] The maltogenic alpha-amylase activity of variants of theinvention towards linear maltodextrins and cyclodextrins may be assayedby measuring the hydrolysis of maltotriose. Hydrolysis is monitored bythe formation of glucose using the GLU-kit (Boehringer Mannheim,Indianapolis Ind.). Hydrolysis of longer maltodextrins, such asmalto-tetraose to -heptaose) and cyclodextrins is monitored by theformation of free reducing ends which is measuredspectrophotometrically.

[0128] Alternatively, amylolytic activity can be assayed using thePhadebas method (BioRad, Inc., Richmond, Calif.) in which the substrateis a water-insoluble cross-linked starch polymer carrying a blue dye(Phadebas Amylase Test) that is hydrolyzed by amylolytic activity toform water-soluble blue fragments which can then be quantitatedspectrophotometrically.

[0129] In cases where the variants of the invention have been altered inthe substrate binding site, it may be desirable to determine whethersuch variant is capable of performing a transglycosylation reaction,which is described below in Example 1, as is normally observed forCGTases.

[0130] Substrate specificity of maltogenic alpha-amylase variants may beassayed by measuring the degree to which such enzymes are capable ofdegrading starch that has been exhaustively treated with theexoglycosylase β-amylase. To screen for variants which show patterns ofdegradation on such a substrate differing from the patterns produced bythe parent CGTase the following assay is performed: β-limit dextrin isprepared by incubating 25 ml 1% amylopectin in Mcllvane buffer (48.5 mMcitrate and 193 mM sodium phosphate pH 5.0) with 24 μg/ml β-amylaseovernight at 30° C. Unhydrolysed amylopectin (i.e., β-limit dextrin) isprecipitated with 1 volume 98% ethanol, washed and redissolved in water.1 ml β-limit dextrin is incubated with 18 μl enzymes (at 2.2 mg/ml) and100 μl 0.2 M citrate-phosphate pH 5.0 for 2 hrs at 30° C. and analysedby HPLC as described above. Total hydrolysis of β-limit dextrin iscarried out in 2M HCI at 95° C. The concentration of reducing ends ismeasured by methods known in the art.

Industrial Applications

[0131] The maltogenic alpha-amylase variants of the invention possessvaluable properties which may be advantageously used in variousindustrial applications. In particular, the enzyme finds potentialapplication for retarding or preventing retrogradation, and thus thestaling, of starch based food common in the baking industry.

[0132] The variant may be used for the preparation of bread and otherbread products in accordance with conventional techniques known in theart.

[0133] It is believed that the modification of the starch fraction byuse of the present invention results in increased volume in bakedproducts and improved organoleptic qualities, such as flavor, mouthfeel, palatability, aroma and crust color.

[0134] The maltogenic alpha-amylase variant may be used as the onlyenzyme or as a major enzymatic activity in combination with one or moreadditional enzymes, such as xylanase, lipase, glucose oxidase and otheroxidoreductases, or an amylolytic enzyme.

[0135] The enzyme variants of the invention also find industrialapplicability as a component in washing, dishwashing and hard-surfacecleaning detergent compositions. Some variants are particularly usefulin a process for the manufacture of linear oligosaccharides, or in theproduction of sweeteners and ethanol from starch, and/or for textiledesizing. Conditions for conventional starch conversion processes,including starch liquefaction and/or saccharification processes, aredescribed in, e.g., U.S. Pat. No. 3,912,590 and in EP patentpublications Nos. 252,730 and 63,909.

[0136] The variants of the invention also find application in processesfor the manufacture of cyclodextrins for various industrialapplications, particularly in the food, cosmetic, chemical, agrochemicaland pharmaceutical industries.

[0137] Therefore in another aspect the invention provides maltogenicalpha-amylase variants for use in a process for the manufacture ofcyclodextrins, in particular α-, β-, γ-, δ-, ε-, and/or ζ-cyclodextrins.In a more preferred embodiment, the invention provides maltogenicalpha-amylase variants for use in a process for the manufacture of α-,β- and γ-cyclodextrins, or mixtures hereof.

[0138] In yet another preferred embodiment, the variants of theinvention may be used for in situ generation of cyclodextrins. In thisway the variants of the invention may be added to a substrate containingmedium in which the enzyme variants are capable of forming the desiredcyclodextrins. This application is particularly well suited for use inmethods of producing baked products as described above, in methods forstabilizing chemical products during their manufacture, and in detergentcompositions.

[0139] Cyclodextrins have an inclusion ability useful for stabilization,solubilization, etc. Thus cyclodextrins can make oxidizing andphotolytic substances stable, volatile substances non-volatile,poorly-soluble substances soluble, and odoriferous substances odorless,etc. and thus are useful to encapsulate perfumes, vitamins, dyes,pharmaceuticals, pesticides and fungicides. Cyclodextrins are alsocapable of binding lipophilic substances such as cholesterol, to removethem from egg yolk, butter, etc.

[0140] Cyclodextrins also find utilization in products and processesrelating to plastics and rubber, where they have been used for differentpurposes in plastic laminates, films, membranes, etc. Also,cyclodextrins have been used for the manufacture of biodegradableplastics.

EXAMPLES

[0141] The invention is further illustrated with reference to thefollowing examples which are not intended to be in any way limiting tothe scope of the invention as claimed.

Example 1 Construction of Variants of Thermoanaerobacter CGTase withAltered Substrate Specificity

[0142] This example describes the construction of CGTase variants withmodified substrate specificity. The variants are derived from a parentThermoanaerobacter sp. CGTase (i.e. the wild type), obtained asdescribed in WO 89/03421 and WO 96/33267.

Bacterial Strains, Plasmids and Growth Conditions

[0143]Escherichia coli ME32 was used for recombinant DNA manipulations.The variants were expressed in SHA273, a derivative of Bacillus subtilis168 which is apr⁻, npr⁻, amyE⁻, amyR2⁻ and prepared by methods known inthe art. pCA31-wt is a E coli-B. subtilus shuttle vector harboring theparent Thermoanaerobacter CGTase, shown in FIG. 1.

DNA Manipulations

[0144] DNA manipulations and transformation of E. coli were essentiallyas described in Sambrook, J., Fritsch, E. J., and Maniatis, T. (1989)Molecular cloning: a laboratory manual, Cold Spring Harbor LaboratoryPress, New York. B. subtilis was transformed using methods known in theart.

Site-directed Mutagenesis

[0145] Mutant CGTase genes were constructed via SOE-PCR method (Nelsonand Long, op.cit.) using the Pwo DNA polymerase (Boehringer Mannheim,Indianapolis, Ind.). The primary PCR reactions were carried out with themutagenesis primers 1 and 2 (SEQ ID NO: 3 and 4) plus an upstream or adownstream primer (SEQ ID NO: 5 or 6) on the template strand,respectively. The reaction products were subsequently used as templatein a second PCR reaction together with the upstream and downstreamprimers. The product of the last reaction was digested with Styl andSpel, and exchanged with the corresponding fragment (640 bp) from thevector pCA31-wt or pCA31-Δ(87-94)(T-CGTase+L82H+D84*+T84bG+F84cY+G84d*+G85*+S86*). The resulting variantplasmids were transformed into E. coli ME32 and vector DNA was purifiedfrom E. coli colonies using the DNA-purification kit from QIAGEN(Qiagen, Inc. Germany). The mutant vectors were finally transformed intoB. subtilis SHA273 for enzyme expression.

[0146] The degeneration of mutagenesis primer 2 (SEQ ID NO: 4)containing A or C/G gave rise to two different amino acid sequences.Thus, two variants of a parent CGTase were constructed. Successfulmutations resulted in restriction sites (Sac II) at positions 7-12 ofSEQ ID NO: 3 and positions 2-7 of SEQ ID NO: 4, which allowed quickscreening of transformants. Mutations were verified by standard DNAsequencing techniques. The correctness of the Styl-Spel fragmentobtained by PCR was also confirmed by DNA sequencing.

Production and Purification of CGTase Proteins

[0147] Enzymes were produced in transformed SHA273 cells grown inshakeflasks at 30-37° C. in 2*TY media containing 10 μg/l kanamycin.After 68-72 h of growth the culture was pelleted and the supernatantseparated from the cells by centrifugation. After filtration through a0.45 μm nitrocellulose filter, the supernatant was directly applied toan α-cyclodextrin-sepharose-6FF affinity column (Monma et al. 1988Biotechnol. Bioeng. 32, 404-407). After washing the column with 10 mMsodium acetate (pH 5.5), the variants were eluted with the same buffersupplemented with 1% (w/v) (α-cyclodextrin. Purity and molecular weightof the variants obtained were checked by SDS-PAGE. Proteinconcentrations were determined by measuring the absorption at 280 nmusing a theoretical extinction coefficient at 1.74 ml/mg⁻¹/cm⁻¹.

Enzyme Assays

[0148] All assays were performed at pH 6.0 and 37° C. The assay forcyclization activity was performed as described by Penninga et al (1995,Biochemistry 34:3368-3376). Starch liquefying activity was measuredusing the Phadebas Amylase Test kit (Pharmacia A/B, Sweden).Transglycosylation activity was assayed in which 2.2 μM of the variantwas incubated with 200 mM maltotriose at 40° C. in 10 mM NaOAc pH 5.0and 1 mM CaCl₂. At different time intervals, aliquots were analyzed byHPLC to measure formation of different maltodextrins. Analyticalseparations of maltodextrins were performed on a Dionex CarboPacPA1-column connected to a Beckman Gold HPLC-system and apulsed-amperometric detector. The gradient was 0-600 mM NaOAc over 15minutes in 0.1 M NaOH. Transglycosylation activity was detected as anincrease in the size of the from three to greater than three glucoseunits covalently linked.

Example 2 Specific PCR Amplification Using Novamyl-specific PCR Primers

[0149] Comparison of Novamyl with CGTases reveals that it is thus farunique in one structural feature: the insertion of a 5 amino acid “loop”in domain A, residues 190 to 194 in the amino acid sequence shown in SEQID NO: 1, that affects the enzyme structure near the active site. It istherefore valuable to have a method of obtaining variants with a similaractive site structure, especially with respect to the Novamyl activesite loop. Here we describe such a method using PCR primers specific tothe Novamyl loop to amplify from natural sources only those clones withthis unique structural feature.

Step 1. PCR Amplification of Glycosylases with Degenerate Primers

[0150] Alignment of amino acid sequences for Novamyl with known CGTasesreveals regions of high homology that can be used to design degenerateoligonucleotide primers for use in the PCR amplification of CGTases froma mixed pool of genomic or cDNA. The resulting fragments of a predictedsize range can then be used as template DNA in further PCRamplifications with Novamyl loop-specific primers as described in Step2.

[0151] Alignment of 10 amino acid sequences most related to Novamyl wasused to identify two regions of high local homology for the design ofdegenerate primers: Primer 1 (SEQ ID NO: 7) corresponding to amino acids88-93 from SEQ ID NO: 1 and Primer 2 (SEQ ID NO: 8) corresponding toamino acids 417-412 from SEQ ID NO: 1.

[0152] Use of these primers on DNA fragments from bacterial sources can,when used as primers in a PCR reaction under standard conditions,amplify a DNA fragment approximately 1,000 basepairs in lengthcontaining the central core of related glycosylases. Resulting PCRfragments could then be used as templates in step 2, as described below.

Step 2. PCR Amplification Using Novamyl Loop-specific Primers (FIG. 3)

[0153] The following primer pair can be used, corresponding to thedegenerate translation of the amino acid sequence in the coding (d3, SEQID 9) or noncoding (d4, SEQ ID NO: 10) DNA strand:

[0154] Primer d3 (SEQ ID NO: 9): degenerate sense primer correspondingto amino acids 190-194 from SEQ ID NO: 1.

[0155] Primer d4 (SEQ ID NO: 10): degenerate anti-sense primercorresponding to amino acids 194-190 from SEQ ID NO: 1.

[0156] Alternatively, it is possible to use the degenerate or exactnucleotide sequence of Novamyl through eight amino acids that containthe Novamyl sequence, Phe188-Thr189-Asp190-Pro191-Ala192-Gly193-Phe194-Ser195 (loop underlined) in bothDNA strands as primers in a PCR reaction:

[0157] Primer d5 (SEQ ID NO: 11): degenerate sense primer correspondingto amino acids 188-193 from SEQ ID NO: 1.

[0158] Primer d6 (SEQ ID NO: 12): degenerate anti-sense primercorresponding to amino acids 195-190 from SEQ ID NO: 1.

[0159] Primer 7 (SEQ ID NO: 13): Exact Novamyl sense primercorresponding to amino acids 188-193 from SEQ ID NO: 1.

[0160] Primer 8 (SEQ ID NO: 14): Exact Novamyl anti-sense primercorresponding to amino acids 195-190 from SEQ ID NO: 1.

[0161] Using the PCR products of step 1 as templates in a PCR reactiontogether with primer pairs 1 and d4, 2 and d4, 1 and d5, 2 and d6, 1 and7, or 2 and 8, only those DNA sequences encoding the Novamyl loop areexpected to produce DNA fragments of approximately the predicted size(FIG. 1, step 2.). Templates lacking the loop-encoding DNA will notproduce a product under standard PCR conditions with an annealingtemperature of 58° C. approximate size of Product Primer pair product A11 + d4 321 A2 1 + d6 324 A3 1 + 8 324 B1 2 + d3 684 B2 2 + d5 690 B3 2 +7 690

Step 3. Reconstruction of Full-length Fragments

[0162] Step 2 yields partial coding sequences of glycosylases thatcontain the Novamyl loop at either the 3′ (A fragments) or 5′ ends (Bfragments) of the DNA fragments. Reassembly of longer clones containingthe loop will require the combining of the A and B fragments by SOE-PCRmethods known in the art.

Example 3 Conversion of CGTases into Novamyl-like enzymes by RandomRe-combination

[0163] In this example, the unique active site loop was used to selecthybrid enzymes with maltogenic alpha-amylase activity from a library ofrandom recombinants. In this method, Novamyl and the cyclic maltodextringlucosyl transferase (CGTase) from Bacillus circulans, were randomlyrecombined by the DNA shuffling method of Crameri A, et al., op.cit.Those resulting mutants containing the Novamyl loop were selected usingPCR as described above in Example 2.

Step 1. PCR Amplification and Shuffling of Novamyl and CGTase (FIG. 2)

[0164] Specific oligonucleotide primers specific for either the Novamylcoding sequence or the CGTase coding sequenced were designed as shown inSEQ ID NO: 15-20.

[0165] The entire Novamyl coding sequence (lacking the signal sequence)was amplified using the Novamyl-specific primer pair #9 and #10 (SEQ IDNO: 15 and 16). Similarly, the mature CGTase coding sequence wasamplified using the CGTase-specific primer pair #11 and #12 (SEQ ID NO:17 and 18). Both amplifications were performed using the followingreaction conditions: 100 μM each primer, 0.2 mM each of dATP, dCTP,dGTP, and TTP, 2.5 U AmpliTaq polymerase (Perkin Elmer, Inc.), and1×concentration of the buffer supplied by the manufacturer. PCR wasperformed in a Perkin Elmer Thermocycler, model 2400, with the followingconditions: 5 minutes at 94 C. 25 cycles of 30 seconds at 94 C., 1minute at 58 C., and 2 minutes at 72 C. followed by a final incubationof 7 minutes at 72° C.

[0166] The resulting PCR products were then subjected to DNA shufflingas described by Stemmer et al. Briefly, the two DNA fragments were mixedin equimolar amounts and randomly digested using DNase I treatment togenerate gene fragments of between 50 and 500 bp. These gene fragmentswere then allowed to anneal to one another and extend in a PCR reactionunder low stringency conditions, resulting in a re-assembling of anintact gene pool containing the reassemble parental DNAs as well aschimeras between the parents. Final amplification of shuffled productswas performed using the general primer pair #13 and #14 (SEQ ID NO: 19and 20) using the PCR conditions described above. Using these primers,all full-length species, both parental and chimeric, were amplified.

Step 2. PCR Amplification Using Novamyl Loop-specific Primers (FIG. 3)

[0167] Those genes within this mixture containing the Novamyl loop werethen selected for as described in Example 2 using the loop-specificprimers. In the first round of amplification, the 5′ and 3′ ends of thegenes containing the loop were amplified by using the general primers incombination with the loop specific primers #7 and #8 (SEQ ID NO: 13 and14) to amplify either the 5′ ends of the genes extending to theloop-encoding sequence #13 and #8 (SEQ ID NO: 19 and 14) or the sequenceextending from the loop to the 3′ ends of the genes #7 and #14 (SEQ IDNO: 13 and 20). As described in Example 2, the resulting fragments werethen assembled using SOE PCR to create full-length genes. These productswere then selectively amplified using primer pairs lacking aNovamyl-specific primer to produce only chimeras: #11, #10 and #12 (SEQID NO: 17, 16 and 18) or #10, #9 and #11 (SEQ ID NO: 16, 15 and 17). Inthis way, only those clones that contained either the CGTase 5′ end andthe Novamyl loop or the CGTase 3′ end and the Novamyl loop wereselected.

[0168] The final PCR product were then digested with the enzymes Xba Iand Mlu I and inserted into a vector containing an intact signalsequence. The resulting clones were transformed into Bacillus, and theresulting polypeptides were sequenced.

[0169] 3 polypeptides obtained by this method were found to be hybridscontaining an N-terminal sequence from Novamyl and a C-terminal sequencefrom the B. circulans CGTase as follows:

[0170] Novamyl amino acids 1-196+CGTase amino acids 198-685

[0171] Novamyl amino acids 1-230+CGTase amino acids 232-685

[0172] Novamyl amino acids 1-590+CGTase amino acids 596-685.

[0173] These results demonstrate that the method is effective forgenerating and selecting hybrids containing the

Example 4 Construction of a Variant of Novamyl with CGTase Activity

[0174] A variant of Novamyl was constructed that has an alteredsubstrate specificity relative to the parent enzyme, in which thevariant has a CGTase-like transglycosylation/cyclization activity notdetectable in the parent enzyme. The variant differs from the parentNovamyl with the amino acid sequence shown in amino acids 1-686 of SEQID NO: 1 in that residues 191-195 were removed, Phel88 was substitutedwith Leu and Thrl89 was substituted with Tyr, termed Δ(191-195)-F188L-T189Y. The variant was constructed by sequence overlapextension PCR (SOE PCR) essentially as described by Nelson and Long(op.cit.). SOE PCR consists of two primary PCRs that produce twooverlapping PCR fragments, both bearing the same modification(s). In asecond round of PCR, the two products from the two primary PCRs aremixed without addition of template DNA.

Oligonucleotide Primers Used in the Construction of Δ(191-195)-F188L-T189Y

[0175] Mutagenic Primer 1 (SEQ ID NO: 21) and Primer 2 (SEQ ID NO: 22).Positions 16-21 of SEQ ID NO: 21 and positions 4-9 of SEQ ID NO: 22 arerestriction sites.

[0176] The Δ (191-195)-F188L-T189Y were obtained using oligomers A82(SEQ ID NO: 23) and B346 (SEQ ID NO: 24) as end-primers.

[0177] DNA manipulations, transformation of Bacillus subtilis, andpurification of the resulting variant was performed as described abovein Example 4. The final purified variant was analysed for the ability toform cyclodextrin from linear starch and compared to the parent Novamylenzyme as described below.

Detection of β-cyclodextrin

[0178] The variant and the parent Novamyl enzyme were diluted with 10 mMcitrate buffer pH 6.0 in order to obtain an equivalent proteinconcentration prior to assay.

[0179] The cyclisation reaction mixture in a final volume of 1 mlcontained:

[0180] 0-50 μl enzyme or variant diluted in 10 mM sodium citrate bufferpH 6.0

[0181] 500 μl 10%(w/v) Paselli SA2 (AVEBE, Foxhol, The Netherlands)dissolved in 10 mM citrate buffer pH 6.0 for a final solution of 5%

[0182] The reaction mixture was pre-incubated in a 50° C. water bath for10 min. before adding the enzyme or variant, and at one-min timeintervals a 100 μl sample was put on ice for further analysis.

[0183] β-cyclodextrin was quantitated on the basis of formation of astable colourless inclusion complex with phenolphthalein; thus, thecolour of the solution decreases with as the amount of β-cyclodextrindetected increases.

[0184] To each of the 100 μl samples from the cyclization reaction 900μl of a working solution (3 ml of 3.75 mM phenolphthalein added to 100ml 0.2 M Na₂CO₃, pH 9.7) was added, and the absorbance immediately readat 552 nm. β-cyclodextrin was quantitated on the basis of a calibrationcurve prepared in a final volume of one-ml as follows:

[0185] 0-50 μl 2 mM β-cyclodextrin (0-100 nmol)

[0186] 50-0 μl milli-Q water

[0187] 900 μl working solution

[0188] 50 μl 10% Paselli SA2

[0189] A new calibration curve was made for each new preparation ofPaselli SA2 solution.

[0190] The results of the cyclization assay are presented in the tablebelow as β-cyclodextrin formation (mmol/mg enzyme) for the variant Δ(191-195)-F188L-T189Y and for the parent enzyme, Novamyl: Time (min)Variant Novamyl 0  0 0 2 160 0 3 230 0 4 240 0 5 320 0 6 380 0 7 390 0 8500 0 10  680 0

[0191] The results clearly demonstrate that the variant, unlike theparent Novamyl enzyme, can form β-cyclodextrin.

Example 5 Construction of a CGTase Variant with Ability to Form LinearOligo-saccharides

[0192] This example describes the construction of a CGTase variantderived from a parent Thermoanaerobacter CGTase.

[0193] Mutant CGTase genes were constructed via SOE-PCR method asdescribed in Example 1. The primary PCR reactions were carried out withthe mutagenesis primers A91 (SEQ ID NO: 26) and A90 (SEQ ID NO: 25) plusan upstream or a downstream primer (SEQ ID NO 5 or 6) on the templatestrand, respectively. The product of the last reaction was digested withBst1107 I and Pst I, and exchanged with the corresponding fragment (250bp) from the vector pCA31-wt or pCA31-(T-CGTase+F189L+*190D+*191P+*192A+*193G+*194F+D195S). Successful mutations resulted in restrictionsites (Xma I) at positions 4-9 of A91 (SEQ ID NO: 26) and positions11-16 of A90 (SEQ ID NO: 25), which allowed quick screening oftransformants. The following mutations were verified by standard DNAsequencing techniques:

[0194] Y260F+L261G+G262D+T263D+N264P+E265G+V266T+*266aA+*266bN+D267H+P268V

[0195]*194aT+*194bD+*194cP+*194dA+*194eG+D196S+Y260F+L261G+G262D+T263D+N264P+E265G+V266T+*266aA+*266bN+D267H+P268V

Example 6 Properties of CGTase Variant with Ability to Form LinearOligosaccharides Inhibition of Starch Retrogradation

[0196] The first variant prepared in Example 5 was tested for itsability to inhibit starch retrogradation was tested as follows:

[0197] 730 mg of 50% (w/w) amylopectin slurry in 0.1 M sodium acetate,at a selected pH (3.7, 4.3 or 5.5) was mixed with 20 μl of an enzymesample, and the mixture was incubated in a sealed ampoule for 1 hour at40° C., followed by incubation at 100° C. for 1 hour in order togelatinize the samples. The sample was then aged for 7 days at roomtemperature to allow recrystallization of the amylopectin. A controlwithout enzyme was included.

[0198] After aging, DSC was performed on the sample by scanning from 5°C. to 95° C. at a constant scan rate of 90° C./hour. The area under thefirst endothermic peak in the thermogram was taken to represent theamount of retrograded amylopectin, and the relative inhibition ofretrogradation was taken as the area reduction (in %) relative to thecontrol without enzyme.

[0199] The result was a relative inhibition of 21%.

Reaction Pattern with Starch

[0200] The variant was compared with Novamyl and with ThermoanaerobacterCGTase by determining the reaction products formed after 24 hoursincubation in 5% (w/v) amylopectin using 50 mM sodium acetate, 1 mMCaCl2, pH 5.0 at 50° C. The reaction products (in % by weight) wereidentified and quantitated using HPLC. Oligosaccharide Novamyl CGTaseVariant  G10 — — 0.7 G9 — — 1.5 G8 — — 2.4 G7 — — 1.9 G6/α-CD — 53.923.1 G5 — — 6.1 G4 — — 8.1 G3/γ-CD — 12.0 14.5 G2 979 − 11.5 G1 2.1 —6.8 β-CD 34.1 23.1

[0201] The results show clearly that whereas the parent CGTaseexclusively forms cyclodextrins, the reaction pattern of the variant hasbeen changed to form both cyclodextrins and linear maltodextrins asinitial products.

1 31 1 2160 DNA Bacillus sp. CDS (1)..(2157) 1 atg aaa aag aaa acg ctttct tta ttt gtg gga ctg atg ctc ctc atc 48 Met Lys Lys Lys Thr Leu SerLeu Phe Val Gly Leu Met Leu Leu Ile -30 -25 -20 ggt ctt ctg ttc agc ggttct ctt ccg tac aat cca aac gcc gct gaa 96 Gly Leu Leu Phe Ser Gly SerLeu Pro Tyr Asn Pro Asn Ala Ala Glu -15 -10 -5 gcc agc agt tcc gca agcgtc aaa ggg gac gtg att tac cag att atc 144 Ala Ser Ser Ser Ala Ser ValLys Gly Asp Val Ile Tyr Gln Ile Ile -1 1 5 10 15 att gac cgg ttt tac gatggg gac acg acg aac aac aat cct gcc aaa 192 Ile Asp Arg Phe Tyr Asp GlyAsp Thr Thr Asn Asn Asn Pro Ala Lys 20 25 30 agt tat gga ctt tac gat ccgacc aaa tcg aag tgg aaa atg tat tgg 240 Ser Tyr Gly Leu Tyr Asp Pro ThrLys Ser Lys Trp Lys Met Tyr Trp 35 40 45 ggc ggg gat ctg gag ggg gtt cgtcaa aaa ctt cct tat ctt aaa cag 288 Gly Gly Asp Leu Glu Gly Val Arg GlnLys Leu Pro Tyr Leu Lys Gln 50 55 60 ctg ggc gta acg aca atc tgg ttg tccccg gtt ttg gac aat ctg gat 336 Leu Gly Val Thr Thr Ile Trp Leu Ser ProVal Leu Asp Asn Leu Asp 65 70 75 aca ctg gcg ggc acc gat aac acg ggc tatcac gga tac tgg acg cgc 384 Thr Leu Ala Gly Thr Asp Asn Thr Gly Tyr HisGly Tyr Trp Thr Arg 80 85 90 95 gat ttt aaa cag att gag gaa cat ttc gggaat tgg acc aca ttt gac 432 Asp Phe Lys Gln Ile Glu Glu His Phe Gly AsnTrp Thr Thr Phe Asp 100 105 110 acg ttg gtc aat gat gct cac caa aac ggaatc aag gtg att gtc gac 480 Thr Leu Val Asn Asp Ala His Gln Asn Gly IleLys Val Ile Val Asp 115 120 125 ttt gtg ccc aat cat tcg act cct ttt aaggca aac gat tcc acc ttt 528 Phe Val Pro Asn His Ser Thr Pro Phe Lys AlaAsn Asp Ser Thr Phe 130 135 140 gcg gaa ggc ggc gcc ctc tac aac aat ggaacc tat atg ggc aat tat 576 Ala Glu Gly Gly Ala Leu Tyr Asn Asn Gly ThrTyr Met Gly Asn Tyr 145 150 155 ttt gat gac gca aca aaa ggg tac ttc caccat aat ggg gac atc agc 624 Phe Asp Asp Ala Thr Lys Gly Tyr Phe His HisAsn Gly Asp Ile Ser 160 165 170 175 aac tgg gac gac cgg tac gag gcg caatgg aaa aac ttc acg gat cca 672 Asn Trp Asp Asp Arg Tyr Glu Ala Gln TrpLys Asn Phe Thr Asp Pro 180 185 190 gcc ggt ttc tcg ctt gcc gat ttg tcgcag gaa aat ggc acg att gct 720 Ala Gly Phe Ser Leu Ala Asp Leu Ser GlnGlu Asn Gly Thr Ile Ala 195 200 205 caa tac ctg acc gat gcg gcg gtt caattg gta gca cat gga gcg gat 768 Gln Tyr Leu Thr Asp Ala Ala Val Gln LeuVal Ala His Gly Ala Asp 210 215 220 ggt ttg cgg att gat gcg gtg aag catttt aat tcg ggg ttc tcc aaa 816 Gly Leu Arg Ile Asp Ala Val Lys His PheAsn Ser Gly Phe Ser Lys 225 230 235 tcg ttg gcc gat aaa ctg tac caa aagaaa gac att ttc ctg gtg ggg 864 Ser Leu Ala Asp Lys Leu Tyr Gln Lys LysAsp Ile Phe Leu Val Gly 240 245 250 255 gaa tgg tac gga gat gac ccc ggaaca gcc aat cat ctg gaa aag gtc 912 Glu Trp Tyr Gly Asp Asp Pro Gly ThrAla Asn His Leu Glu Lys Val 260 265 270 cgg tac gcc aac aac agc ggt gtcaat gtg ctg gat ttt gat ctc aac 960 Arg Tyr Ala Asn Asn Ser Gly Val AsnVal Leu Asp Phe Asp Leu Asn 275 280 285 acg gtg att cga aat gtg ttc ggcaca ttt acg caa acg atg tac gat 1008 Thr Val Ile Arg Asn Val Phe Gly ThrPhe Thr Gln Thr Met Tyr Asp 290 295 300 ctt aac aat atg gtg aac caa acgggg aac gag tac aaa tac aaa gaa 1056 Leu Asn Asn Met Val Asn Gln Thr GlyAsn Glu Tyr Lys Tyr Lys Glu 305 310 315 aat cta atc aca ttt atc gat aaccat gat atg tca aga ttt ctt tcg 1104 Asn Leu Ile Thr Phe Ile Asp Asn HisAsp Met Ser Arg Phe Leu Ser 320 325 330 335 gta aat tcg aac aag gcg aatttg cac cag gcg ctt gct ttc att ctc 1152 Val Asn Ser Asn Lys Ala Asn LeuHis Gln Ala Leu Ala Phe Ile Leu 340 345 350 act tcg cgg ggt acg ccc tccatc tat tat gga acc gaa caa tac atg 1200 Thr Ser Arg Gly Thr Pro Ser IleTyr Tyr Gly Thr Glu Gln Tyr Met 355 360 365 gca ggc ggc aat gac ccg tacaac cgg ggg atg atg ccg gcg ttt gat 1248 Ala Gly Gly Asn Asp Pro Tyr AsnArg Gly Met Met Pro Ala Phe Asp 370 375 380 acg aca acc acc gcc ttt aaagag gtg tca act ctg gcg ggg ttg cgc 1296 Thr Thr Thr Thr Ala Phe Lys GluVal Ser Thr Leu Ala Gly Leu Arg 385 390 395 agg aac aat gcg gcg atc cagtac ggc acc acc acc cag cgt tgg atc 1344 Arg Asn Asn Ala Ala Ile Gln TyrGly Thr Thr Thr Gln Arg Trp Ile 400 405 410 415 aac aat gat gtt tac atttat gaa cgg aaa ttt ttc aac gat gtc gtg 1392 Asn Asn Asp Val Tyr Ile TyrGlu Arg Lys Phe Phe Asn Asp Val Val 420 425 430 ttg gtg gcc atc aat cgaaac acg caa tcc tcc tat tcg att tcc ggt 1440 Leu Val Ala Ile Asn Arg AsnThr Gln Ser Ser Tyr Ser Ile Ser Gly 435 440 445 ttg cag acg gcc ttg ccaaat ggc agc tat gcg gat tat ctg tca ggg 1488 Leu Gln Thr Ala Leu Pro AsnGly Ser Tyr Ala Asp Tyr Leu Ser Gly 450 455 460 ctg ttg ggg ggg aac gggatt tcc gtt tcc aat gga agt gtc gct tcg 1536 Leu Leu Gly Gly Asn Gly IleSer Val Ser Asn Gly Ser Val Ala Ser 465 470 475 ttc acg ctt gcg cct ggagcc gtg tct gtt tgg cag tac agc aca tcc 1584 Phe Thr Leu Ala Pro Gly AlaVal Ser Val Trp Gln Tyr Ser Thr Ser 480 485 490 495 gct tca gcg ccg caaatc gga tcg gtt gct cca aat atg ggg att ccg 1632 Ala Ser Ala Pro Gln IleGly Ser Val Ala Pro Asn Met Gly Ile Pro 500 505 510 ggt aat gtg gtc acgatc gac ggg aaa ggt ttt ggg acg acg cag gga 1680 Gly Asn Val Val Thr IleAsp Gly Lys Gly Phe Gly Thr Thr Gln Gly 515 520 525 acc gtg aca ttt ggcgga gtg aca gcg act gtg aaa tcc tgg aca tcc 1728 Thr Val Thr Phe Gly GlyVal Thr Ala Thr Val Lys Ser Trp Thr Ser 530 535 540 aat cgg att gaa gtgtac gtt ccc aac atg gcc gcc ggg ctg acc gat 1776 Asn Arg Ile Glu Val TyrVal Pro Asn Met Ala Ala Gly Leu Thr Asp 545 550 555 gtg aaa gtc acc gcgggt gga gtt tcc agc aat ctg tat tct tac aat 1824 Val Lys Val Thr Ala GlyGly Val Ser Ser Asn Leu Tyr Ser Tyr Asn 560 565 570 575 att ttg agt ggaacg cag aca tcg gtt gtg ttt act gtg aaa agt gcg 1872 Ile Leu Ser Gly ThrGln Thr Ser Val Val Phe Thr Val Lys Ser Ala 580 585 590 cct ccg acc aacctg ggg gat aag att tac ctg acg ggc aac ata ccg 1920 Pro Pro Thr Asn LeuGly Asp Lys Ile Tyr Leu Thr Gly Asn Ile Pro 595 600 605 gaa ttg ggg aattgg agc acg gat acg agc gga gcc gtt aac aat gcg 1968 Glu Leu Gly Asn TrpSer Thr Asp Thr Ser Gly Ala Val Asn Asn Ala 610 615 620 caa ggg ccc ctgctc gcg ccc aat tat ccg gat tgg ttt tat gta ttc 2016 Gln Gly Pro Leu LeuAla Pro Asn Tyr Pro Asp Trp Phe Tyr Val Phe 625 630 635 agc gtt cca gcagga aag acg att caa ttc aag ttc ttc atc aag cgt 2064 Ser Val Pro Ala GlyLys Thr Ile Gln Phe Lys Phe Phe Ile Lys Arg 640 645 650 655 gcg gat ggaacg att caa tgg gag aat ggt tcg aac cac gtg gcc aca 2112 Ala Asp Gly ThrIle Gln Trp Glu Asn Gly Ser Asn His Val Ala Thr 660 665 670 act ccc acgggt gca acc ggt aac att act gtt acg tgg caa aac tag 2160 Thr Pro Thr GlyAla Thr Gly Asn Ile Thr Val Thr Trp Gln Asn 675 680 685 2 719 PRTBacillus sp. 2 Met Lys Lys Lys Thr Leu Ser Leu Phe Val Gly Leu Met LeuLeu Ile -30 -25 -20 Gly Leu Leu Phe Ser Gly Ser Leu Pro Tyr Asn Pro AsnAla Ala Glu -15 -10 -5 Ala Ser Ser Ser Ala Ser Val Lys Gly Asp Val IleTyr Gln Ile Ile -1 1 5 10 15 Ile Asp Arg Phe Tyr Asp Gly Asp Thr Thr AsnAsn Asn Pro Ala Lys 20 25 30 Ser Tyr Gly Leu Tyr Asp Pro Thr Lys Ser LysTrp Lys Met Tyr Trp 35 40 45 Gly Gly Asp Leu Glu Gly Val Arg Gln Lys LeuPro Tyr Leu Lys Gln 50 55 60 Leu Gly Val Thr Thr Ile Trp Leu Ser Pro ValLeu Asp Asn Leu Asp 65 70 75 Thr Leu Ala Gly Thr Asp Asn Thr Gly Tyr HisGly Tyr Trp Thr Arg 80 85 90 95 Asp Phe Lys Gln Ile Glu Glu His Phe GlyAsn Trp Thr Thr Phe Asp 100 105 110 Thr Leu Val Asn Asp Ala His Gln AsnGly Ile Lys Val Ile Val Asp 115 120 125 Phe Val Pro Asn His Ser Thr ProPhe Lys Ala Asn Asp Ser Thr Phe 130 135 140 Ala Glu Gly Gly Ala Leu TyrAsn Asn Gly Thr Tyr Met Gly Asn Tyr 145 150 155 Phe Asp Asp Ala Thr LysGly Tyr Phe His His Asn Gly Asp Ile Ser 160 165 170 175 Asn Trp Asp AspArg Tyr Glu Ala Gln Trp Lys Asn Phe Thr Asp Pro 180 185 190 Ala Gly PheSer Leu Ala Asp Leu Ser Gln Glu Asn Gly Thr Ile Ala 195 200 205 Gln TyrLeu Thr Asp Ala Ala Val Gln Leu Val Ala His Gly Ala Asp 210 215 220 GlyLeu Arg Ile Asp Ala Val Lys His Phe Asn Ser Gly Phe Ser Lys 225 230 235Ser Leu Ala Asp Lys Leu Tyr Gln Lys Lys Asp Ile Phe Leu Val Gly 240 245250 255 Glu Trp Tyr Gly Asp Asp Pro Gly Thr Ala Asn His Leu Glu Lys Val260 265 270 Arg Tyr Ala Asn Asn Ser Gly Val Asn Val Leu Asp Phe Asp LeuAsn 275 280 285 Thr Val Ile Arg Asn Val Phe Gly Thr Phe Thr Gln Thr MetTyr Asp 290 295 300 Leu Asn Asn Met Val Asn Gln Thr Gly Asn Glu Tyr LysTyr Lys Glu 305 310 315 Asn Leu Ile Thr Phe Ile Asp Asn His Asp Met SerArg Phe Leu Ser 320 325 330 335 Val Asn Ser Asn Lys Ala Asn Leu His GlnAla Leu Ala Phe Ile Leu 340 345 350 Thr Ser Arg Gly Thr Pro Ser Ile TyrTyr Gly Thr Glu Gln Tyr Met 355 360 365 Ala Gly Gly Asn Asp Pro Tyr AsnArg Gly Met Met Pro Ala Phe Asp 370 375 380 Thr Thr Thr Thr Ala Phe LysGlu Val Ser Thr Leu Ala Gly Leu Arg 385 390 395 Arg Asn Asn Ala Ala IleGln Tyr Gly Thr Thr Thr Gln Arg Trp Ile 400 405 410 415 Asn Asn Asp ValTyr Ile Tyr Glu Arg Lys Phe Phe Asn Asp Val Val 420 425 430 Leu Val AlaIle Asn Arg Asn Thr Gln Ser Ser Tyr Ser Ile Ser Gly 435 440 445 Leu GlnThr Ala Leu Pro Asn Gly Ser Tyr Ala Asp Tyr Leu Ser Gly 450 455 460 LeuLeu Gly Gly Asn Gly Ile Ser Val Ser Asn Gly Ser Val Ala Ser 465 470 475Phe Thr Leu Ala Pro Gly Ala Val Ser Val Trp Gln Tyr Ser Thr Ser 480 485490 495 Ala Ser Ala Pro Gln Ile Gly Ser Val Ala Pro Asn Met Gly Ile Pro500 505 510 Gly Asn Val Val Thr Ile Asp Gly Lys Gly Phe Gly Thr Thr GlnGly 515 520 525 Thr Val Thr Phe Gly Gly Val Thr Ala Thr Val Lys Ser TrpThr Ser 530 535 540 Asn Arg Ile Glu Val Tyr Val Pro Asn Met Ala Ala GlyLeu Thr Asp 545 550 555 Val Lys Val Thr Ala Gly Gly Val Ser Ser Asn LeuTyr Ser Tyr Asn 560 565 570 575 Ile Leu Ser Gly Thr Gln Thr Ser Val ValPhe Thr Val Lys Ser Ala 580 585 590 Pro Pro Thr Asn Leu Gly Asp Lys IleTyr Leu Thr Gly Asn Ile Pro 595 600 605 Glu Leu Gly Asn Trp Ser Thr AspThr Ser Gly Ala Val Asn Asn Ala 610 615 620 Gln Gly Pro Leu Leu Ala ProAsn Tyr Pro Asp Trp Phe Tyr Val Phe 625 630 635 Ser Val Pro Ala Gly LysThr Ile Gln Phe Lys Phe Phe Ile Lys Arg 640 645 650 655 Ala Asp Gly ThrIle Gln Trp Glu Asn Gly Ser Asn His Val Ala Thr 660 665 670 Thr Pro ThrGly Ala Thr Gly Asn Ile Thr Val Thr Trp Gln Asn 675 680 685 3 39 DNAArtificial Sequence Mutagenisis Primer 1 3 ccgatcccgc gggattctcattagcagatt tagatcagc 39 4 32 DNA Artificial Sequence Mutagenesis primer2 4 cccgcgggat cggtaanatt acggtaaatt cc 32 5 24 DNA Artificial SequenceUpstream Primer 5 tattataagg ggctccatta cctg 24 6 24 DNA ArtificialSequence Downstream Primer 6 cggatacttc agtttccaat gttg 24 7 18 DNAArtificial Sequence Primer 1 7 ksctatcayg ghtactgg 18 8 18 DNA Primer 28 macrtcrttr ttkatcca 18 9 15 DNA Artificial Sequence Primer D3 9gayccngcng gntty 15 10 15 DNA Artificial Sequence Primer D4 10raanccngcn ggrtc 15 11 17 DNA Artificial Sequence Primer D5 11ttyacngayc cngcngg 17 12 17 DNA Artificial Sequence Primer D6 12ccngcnggrt cngtraa 17 13 17 DNA Artificial Sequence Primer 7 13ttcacggatc cagccgg 17 14 17 DNA Artificial Sequence Primer 8 14ccggctggat ccgtgaa 17 15 58 DNA Artificial Sequence Novamyl 5′ primer #915 gattacgcca agcttctaga tgcctgcagc agcagccgta agcagttccg caagcgtc 58 1642 DNA Artificial Sequence Novamyl 3′ primer #10 16 aacactaagctttggacgcg tatccatttc tttgacgttc ca 42 17 58 DNA Artificial SequenceCGTase 5′ primer #11 17 gattacgcca agcttctaga tgcctgcagc agcagccgtagcaccggata cttcagtt 58 18 42 DNA Artificial Sequence CGTase 3′ primer#12 18 aacactaagc tttggacgcg tagacaagtt gtagaagaag gt 42 19 18 DNAArtificial Sequence General 5′ primer #13 19 gattacgcca agcttcta 18 2021 DNA Artificial Sequence General 3′ primer # 14 20 aacactaagctttggacgcg t 21 21 36 DNA Artificial Sequence Mutasgenesis primer 1 21cttgtacgat cttgcagatc tgtcgcagga aaatgg 36 22 41 DNA Artificial SequenceMutagenesis primer 2 22 gacagatctg caagatcgta caagtttctt cattgcgcct c 4123 20 DNA Artificial Sequence A82 Oligomer 23 ggggatctgg agggggttcg 2024 22 DNA Artificial Sequence B346 oligomer 24 tttgtactcg ttccccgttt gg22 25 42 DNA Artificial Sequence Primer A90 25 ggttggcagt cccgggatcgtctccaaacc actcgccaaa tg 42 26 41 DNA Artificial Sequence Primer A91 26gatcccggga ctgccaacca tgtaaataat acgtattttg c 41 27 5 PRT ArtificialSequence Variation 27 Asp Pro Ala Gly Phe 1 5 28 4 PRT ArtificialSequence Variation 28 Asp Ala Gly Phe 1 29 4 PRT Artificial SequenceVariation 29 Asp Pro Gly Phe 1 30 6 PRT Artificial Sequence Variation 30Asp Pro Ala Ala Gly Phe 1 5 31 7 PRT Artificial Sequence Variation 31Asp Pro Ala Ala Gly Gly Phe 1 5

1. A polypeptide which: a) has at least 70% identity to amino acids1-686 of SEQ ID NO: 1; b) comprises an amino acid modification which isan insertion, substitution or deletion compared to SEQ ID NO: 1 in aregion corresponding to amino acids 40-43, 78-85, 136-139, 173-180,189-195 or 259-268; and c) has the ability to form cyclodextrin whenacting on starch.
 2. The polypeptide of claim 1 wherein the modificationcomprises a substitution with or insertion of the amino acid residuepresent at the corresponding position of a cyclodextringlucanotransferase (CGTase) and/or deletion of an amino acid residuewhich is not present at that position in a cyclodextringlucanotransferase (CGTase).
 3. The polypeptide of claim 1 or 2 whereinthe CGTase is derived from a strain of Bacillus, Brevibacterium,Clostridium, Corynebacterium, Klebsiella, Micrococcus,Thermoanaerobacter or Thermoanaerobacterium.
 4. The polypeptide of anyof claims 1-3 wherein the modification comprises a deletion in theregion 190-195, preferably the deletion Δ (191-195).
 5. The polypeptideof any of claims 1-4 which further comprises a substitution of aminoacid 188 and/or 189, preferably F188L and/or Y189Y.
 6. A method ofconstructing a variant of a parent maltogenic alpha-amylase, whichmethod comprises: a) modifying the amino acid sequence by an insertion,substitution or deletion in a region corresponding to amino acids 40-43,78-85, 136-139, 173-180, 189-195 or 259-268 of SEQ ID NO: 1; b)optionally modifying the amino acid sequence by an insertion,substitution or deletion at a position other than a); c) optionallyrepeating a)-b) recursively; d) preparing the variant resulting froma)-c); e) testing the variant by hydrolyzing starch and analyzing theinitial products; f) optionally repeating a)-e) recursively; and g)selecting a variant having the ability to form cyclodextrin from starch.7. A polypeptide which: a) has an amino acid sequence having at least70% identity to a parent cyclodextrin glucanotransferase (CGTase); b)comprises an amino acid modification which is an insertion, substitutionor deletion compared to the parent CGTase in a region corresponding toamino acids 40-43, 78-85, 136-139, 173-180, 189-195 or 259-268 of SEQ IDNO: 1; and c) has the ability to form linear oligosaccharides as aninitial product when acting on starch.
 8. The polypeptide of claim 7wherein the modification comprises a substitution with or insertion ofthe amino acid residue present at the corresponding position of SEQ IDNO: 1 and/or deletion of an amino acid residue which is not present atthat position in SEQ ID NO:
 1. 9. The polypeptide of claim 7 or 8,wherein the modification is an insertion of the amino acid sequenceDPAGF (Asp-Pro-Ala-Gly-Phe) at a position corresponding to D190-F194 ofSEQ ID NO:
 1. 10. The polypeptide of any of claims 7-9, wherein theCGTase is derived from a strain of Bacillus, Brevibacterium,Clostridium, Corynebacterium, Klebsiella, Micro-coccus,Thermoanaerobacter or Thermoanaerobacterium.
 11. The polypeptide of anyof claims 7-10, which further comprises a modification in a regioncorresponding to amino acids 37-39, 44-45, 135, 140-145, 181-186,269-273, or 377-383 of SEQ ID NO:
 1. 12. A method of constructing avariant of a parent cyclodextrin glucanotransferase (CGTase), whichmethod comprises: a) modifying the amino acid sequence by an insertion,substitution or deletion in a region corresponding to amino acids 40-43,78-85, 136-139, 173-180, 189-195 or 259-268 of SEQ ID NO: 1; b)optionally modifying the amino acid sequence by an insertion,substitution or deletion at a position other than a); c) optionallyrepeating a)-b) recursively; d) preparing the variant resulting froma)-c); e) testing the variant by hydrolyzing starch and analyzing theinitial products; f) optionally repeating a)-e) recursively; and g)selecting a variant having the ability to form linear oligosaccharidesas an initial product from starch.
 13. A nucleic acid sequence encodingthe polypeptide of any of claims 1-5 or 7-11, preferably operably linkedto one or more control sequences which direct the expression of thevariant in a suitable expression host.
 14. A recombinant expressionvector comprising the nucleic acid sequence of claim 13, a promoter, andtranscriptional and translational stop signals, and preferably furthercomprising a selectable marker.
 15. A transformed host cell comprisingthe nucleic acid sequence of claim 13 or the vector of claim
 14. 16. Amethod for producing the polypeptide of any of claims 1-5 or 7-11,comprising: a) cultivating a host cell comprising a nucleic acidconstruct comprising a nucleic acid sequence encoding said variant underconditions conducive to expression of the variant; and b) recovering thevariant.
 17. A method for constructing a maltogenic alpha-amylase,comprising: a) recombining DNA encoding a cyclodextringlucanotransferase (CGTase) and DNA encoding a maltogenic alpha-amylase;b) using the recombinant DNA to express a polypeptide; and c) testingthe polypeptide to select a polypeptide having the ability to formlinear oligosaccharides when acting on starch.
 18. The method of claim17, further comprising amplifying the recombinant DNA with primersencoding a partial amino acid sequence of amino acids 1-686 of SEQ IDNO: 1 in combination with primers encoding a partial sequence of theCGTase, said primers preferably comprising at least 5 amino acidresidues, more preferably comprising one or more of positions 188-196,most preferably comprising positions 190-194.
 19. The method of claim 17or 18, wherein the primers encoding a partial sequence of SEQ ID NO: 1comprise a partial sequence of the DNA sequence shown in SEQ ID NO: 1.20. The method of any of claims 17-19, wherein the mature forms of theCGTase and the maltogenic alpha-amylase have at least 40% identity,preferably at least 50%, more preferably at least 60%.
 21. The method ofany of claims 17-20, wherein the CGTase is derived from a strain ofBacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella,Micro-coccus, Thermoanaerobacter or Thermoanaerobacterium.
 22. A methodof selecting DNA encoding maltogenic alpha-amylase in a DNA pool,comprising: a) amplifying DNA encoding maltogenic alpha-amylase by apolymerase chain reaction (PCR) using primers encoding a partial aminoacid sequence of amino acids 1-686 of SEQ ID NO: 1, preferablycomprising at least 5 amino acid residues, preferably comprising one ormore of positions 188-196, more preferably comprising positions 190-194,b) cloning and expressing the amplified DNA, and c) screening formaltogenic alpha-amylase activity.
 23. A process for preparing a doughor a baked product prepared from the dough which comprises adding thepolypeptide of any of claims 7-11 or a variant constructed by the methodof any of claims 12 or 17-20 to the dough in an amount which iseffective to retard the staling of the bread.
 24. A process forpreparing cyclodextrin, comprising treating liquefied starch with thepolypeptide of any of claims 1-5 or the variant constructed by themethod of claim
 6. 25. A method for producing a variant, comprising: a)constructing the variant by the method of claim 6 or 12; b) transforminga microorganism with a DNA sequence encoding the variant; c) cultivatingthe transformed microorganism under conditions which are conducive forproducing the variant, and d) recovering the variant from the resultingculture broth.